Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Method of detaching cells – digesting tissue or establishing...
Patent
1997-05-02
1999-05-25
Saucier, Sandra E.
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Method of detaching cells, digesting tissue or establishing...
C12N 500
Patent
active
059069392
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to an improvement of the methods of collection and manipulation of adherent eukaryotic cells or of cultured tissues.
Cell culture is currently one of the unavoidable procedures entailed in genetic engineering techniques, either for expressing recombinant proteins or, when these cells have been transfected by recombinant viruses, as host cells which can be administered in gene therapy. In the latter case, cells transfected by viral or retroviral vectors are in some cases reimplanted directly in tissues: a review of these different techniques is given in the work entitled "Therapie genique, l'ADN medicaments" coordinated by Axel Kahn, publisher John Libby, (1993); reference may usefully be made also to the paper by R. C. Mulligan, Science (1993), Vol.: 260, p 926.
The yield of viable cells is a critical factor when eukaryotic cells are cultured, both as regards the actual quality of the cells or of the products which are obtained therefrom, and as regards the questions of yield which influence both the amounts of products obtained and the production cost of the said product; in particular when a gene therapy procedure is carried out by injection of cells, for example packaging cells, containing a recombinant virus or retrovirus, the infectious titre of the virus or retrovirus depending directly on the physiological state of the packaging cells and on their concentration.
Adherent cells are often used in cell culture for the production of products which are either secreted by these cells or as such in gene therapy; the most classical examples are either CHO cells (Chinese hamster ovary cells) for the production of recombinant molecules or cloned receptors, or other, fibroblast cells used in gene therapy, and especially cells derived from 3T3 such as NIH-3T3.
In order to detach the cells from their support, various physical techniques (scraper) or chemical techniques (chelators of the EDTA type or proteolytic enzymes of the trypsin type) have been employed. All these techniques have some drawbacks.
The use of physical means or of chelators such as EDTA is intricate and gives rise to the appearance of aggregate as well as a decrease in viability; proteolytic enzymes such as trypsin degrade the cell surface proteins, decrease viability and interfere with their biological property without even completely eliminating the presence of aggregates, leading to lower degrees of infectivity than could be the case; the cells no longer have available to them the surface glycoproteins enabling them to produce retroviruses at an optimum titre.
This applies, for example, to the use of NIH-3T3 packaging cells, which are mouse fibroblasts used in gene therapy, and are cultured on plastic supports and then collected and injected directly into tumours; the principle of this type of therapy is to make particular derivatives of NIH-3T3 cells, known as M11 cells, produce therapeutic retroviruses in vivo within the tumours; as an example of such a treatment, reference may be made to the papers by M. Caruso et al. in Proc. Nat. Acad. Sci. USA, (1993) 90: 7024-7028; or K. W. Culver et al., Science 256 No. 5063 pp. 1550-1551 (1992). The integrity and capacity of these cells to produce viruses are hence fundamental to the therapeutic efficacy of the treatment.
Aggregates have a whole series of drawbacks, the biggest of which are that they give rise to an underestimate of the number of cells and very poor reproducibility of the cell counts. Furthermore, the presence of aggregates interferes with the freezing processes which are essential to good storage of the cells, the cells situated in the middle of the aggregates not being reached by the preservation products such as dimethyl sulphoxide (DMSO), thereby making them more fragile and giving rise to greater mortality. Lastly, aggregates interfere with the diffusion of the cells when they are injected into tissues or tumours in the course of gene therapy treatments.
The devices permitting bulk cell culture of adherent eukaryotic cells are of various types, the
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patent: 5554527 (1996-09-01), Fickenscher
Thurn et al., "Heparin-induced aggregation of lymphoid cells", J. Cell. Physiol. 126 (3) : 352-8 (1986).
Clarke et al., "Attachment of fibroblasts to a polyanionic surface", Exp. Cell Res. 102 (2) :441-5 (1976).
Klebe et al., "Effect of glycosaminoglycans on fibronectin-mediated cell attachment" J. Cell. Physiol. 112 (1) : 5-9 (1982).
San Antonio et al, "Heparin Inhibits the Attachment and Growth of Balb/c-3T3 Fibroblasts on Collagen Substrata", Journal of Cellular Physiology 150(1):8-16 (1992).
Saucier Sandra E.
Universite Pierre Et Marie Curie (Paris VI)
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